Saccharomyces cerevisiae Bpt1p is an ATP-binding cassette (ABC) protein that belongs to the MRP subfamily and is a close homologue of the glutathione conjugate (GS conjugate) transporter Ycf1p. The function of Bpt1p has previously been evaluated only in vitro, by using nonphysiological substrates. In the present study we examined the localization, regulation, and transport properties of Bpt1p in vivo, as well as its capacity to transport a set of prototypical MRP substrates in vitro. Our results show that Bpt1p, like Ycf1p, localizes to the yeast vacuolar membrane, plays a role in cadmium detoxification and ade2 pigmentation in vivo, and can participate in the transport of GS conjugates and glucuronate conjugates, as well as free glutathione, in vitro. However, in all of these cases the contribution of Bpt1p is substantially less than that of Ycf1p. In addition, the expression patterns of YCF1 and BPT1 differ significantly. Whereas YCF1 expression is markedly increased by cadmium, adenine limitation in an ade2 strain, or overexpression of the stress-responsive transcription factor Yap1p, BPT1 expression is only modestly affected under these conditions. Thus, although the functional capabilities of Bpt1p and Ycf1p overlap, their differences in regulation and substrate preference imply that they contribute to cellular detoxification processes in different ways.With the complete genome of numerous organisms now in hand, the ATP-binding cassette (ABC) transporter superfamily has emerged as the largest membrane protein superfamily in both prokaryotes and eukaryotes, including microbes, plants, and animals (10-12, 17, 40, 45). Members of this superfamily catalyze the MgATP-energized transport of a broad range of substrates across biological membranes. Mutational loss of function of ABC proteins has been implicated in an increasing number of inherited diseases (11), and overexpression of certain ABC transporters has been shown to enhance multidrug resistance and the elimination of xenobiotics (1, 2). Consequently, elucidation of the biochemical activity, substrate specificity, and physiological regulation of ABC transporters is of both clinical and general biological significance.Phylogenetic analysis has provided a valuable road map for formulating hypotheses about the function and substrate(s) of a particular transporter. ABC proteins can be divided into seven subfamilies based on sequence relatedness, designated ABCA through ABCG (11, 12, 45; http://nutrigene.4t.com/ humanabc.htm). The emergent view is that members of a particular subfamily are likely to exhibit some degree of overlap in substrate specificity and/or function. This point is well illustrated by members of the human ABCC subfamily, also designated the multidrug resistance-associated protein (MRP) subfamily, several of which participate in cellular detoxification processes.In humans, the ABCC/MRP subfamily contains 12 members, 5 of which (MRP1 through MRP5) are implicated in multidrug transport. Critically, most drugs are either transported by the MRPs ...

The folding of nascent secretory and membrane proteins is monitored by the endoplasmic reticulum (ER) quality control system. Misfolded proteins are retained in the ER and can be removed by ER-associated degradation. As a model for the ER quality control of multispanning membrane proteins in yeast, we have been studying mutant forms of Ste6p. Here, we identify mislocalized mutant forms of Ste6p that induce the formation of, and localize to, prominent structures that are absent in normal cells. We have named these structures ER-associated compartments (ERACs), based on their juxtaposition to and connection with the ER, as observed by fluorescence and electron microscopy. ERACs comprise a network of tubulo-vesicular structures that seem to represent proliferated ER membranes. Resident ER lumenal and membrane proteins are present in ERACs in addition to their normal ER localization, suggesting there is no barrier for their entry into ERACs. However, the forms of Ste6p in ERACs are excluded from the ER and do not enter the secretory pathway; instead, they are ultimately targeted for ER-associated degradation. The presence of ERACs does not adversely affect secretory protein traffic through the ER and does not lead to induction of the unfolded protein response. We propose that ERACs may be holding sites to which misfolded membrane proteins are specifically diverted so as not to interfere with normal cellular functions. We discuss the likelihood that related ER membrane proliferations that form in response to certain other mutant or unassembled membrane proteins may be substantially similar to ERACs. INTRODUCTIONProteins that traffic via the exocytic pathway are translocated into the membrane or lumen of the ER and are then transported to the Golgi complex where sorting to a variety of cellular locations occurs. In addition to specific sequences that promote exit from the ER and delivery to the Golgi (reviewed in Barlowe, 2003), it is clear that for successful vesicular transport, proteins need to be properly folded and in some cases oligomerized. The surveillance system that detects improper folding has been referred to as "ER quality control" and is present in all eukaryotes, including yeast (reviewed in Ellgaard and Helenius, 2003;Kostova and Wolf, 2003). Misfolded proteins are retained in the ER and in certain cases sequestered; however, the mechanisms involved are only partially understood. Proteins that cannot fold properly are generally thought to be retrotranslocated from the ER and subjected to ER-associated degradation (ERAD) by the ubiquitin-proteasome system (reviewed in Hampton, 2002;Jarosch et al., 2003).Although some ER quality control substrates are simply retained in the ER, others induce and localize to proliferated extensions of the ER. Examples of these ER proliferations include structures containing aggregates of misfolded lumenal proteins, called Russell bodies (Valetti et al., 1991;Umebayashi et al., 1997); proliferations of the ER-Golgi intermediate compartment (ERGIC) or vesicular-tubular clus...

Ycf1p is the prototypical member of the yeast multidrug resistance-associated protein (MRP) subfamily of ATP-binding cassette (ABC) transporters. Ycf1p resides in the vacuolar membrane and mediates glutathione-dependent transport processes that result in resistance to cadmium and other xenobiotics. A feature common to many MRP proteins that distinguishes them from other ABC transporters is the presence of a hydrophobic N-terminal extension (NTE), whose function is not clearly established. The NTE contains a membrane spanning domain (MSD0) with five transmembrane spans and a cytosolic linker region (L0). The goal of this study was to determine the functional significance of the NTE of Ycf1p by examining the localization and functional properties of Ycf1p partial molecules, expressed either singly or together. We show that MSD0 plays a critical role in the vacuolar membrane trafficking of Ycf1p, whereas L0 is dispensable for localization. On the other hand, L0 is required for transport function, as determined by monitoring cadmium resistance. We also examine an unusual aspect of Ycf1p biology, namely, the posttranslational proteolytic processing that occurs within a lumenal loop of Ycf1p. Processing is shown to be Pep4p dependent and thus serves as a convenient marker for proper vacuolar localization. The processed fragments associate with each other, suggesting that these natural cleavage products contribute together to Ycf1p function.